Micromechanical measurements in living embryos

点击翻译按钮获取中文摘要

The embryo is a complex system wherein local tissue displacement and deformation is the result of local and distant force-generating mechanisms coupled through the largely-unknown mechanical properties of the composite tissues. One particular case in point is that of neurulation, the process by which the early sheet of cells, called the neural ectoderm, folds itself into the three dimensional structure that is the framework upon which the vertebrate central nervous system grows. At its simplest, such as neurulation in the spinal cord, the process involves the folding of a sheet roughly into a cylinder but even that is poorly understood. Neurulation in the brain is far more complex but essential for us to understand; errors in its morphogenesis are the root cause of debilitating and fatal birth defects. Thanks to novel imaging and image processing technologies, we have made great strides in developing methods to capture the movements of cells and tissues. Three-dimensional time-lapse images, analysed using in toto cell tracking and computational analyses show a rich spectrum of tissue remodelling. However, despite this apparently complex scheme, we believe that these patterns could originate from a well-orchestrated series of stereotypical force-generating mechanisms that are patterned in space and overlapping in their influence. We can already make predictions of how these may act but to verify these models and progress further we need far greater insight into the changing physical properties of tissues as they develop. Biologists are in need of tools to address such problems in the context of the complex and changing conditions that exist within the animal embryo. Our aim is to develop such a tool and use it to study the balance between active processes and the underlying mechanical properties of developing tissues that is essential in shaping correct morphogenesis of the embryo.We plan in this project to develop a minimally-invasive tool able to probe the local mechanical response of living tissues. The device will be relatively portable, mountable on a standard microscope stage. It will impose a controlled force upon a ferromagnetic bead located in the biological sample. The direction and magnitude of force to be controlled. Our preliminary tests have demonstrated that such an experiment is achievable in zebrafish embryos. Animals develop normally with these particles in place and modest magnetic fields can be used to gently displace beads within these embryos. This methodology will enable us to investigate largely unexplored areas of developmental biology. First, we will characterise for the the elastic and viscous/plastic properties of living tissues within a normally developing embryo. This information is important to establish the range of forces required to observed processes, and to discriminates between possible mechanisms. We will focus our attention to the analysis of tissue maturation during development. The transition from blastula to gastrula is a good example where cells thought to progressively form tighter junctions. We will follow the evolution of the tissue mechanical properties with developmental time and ask if this temporal variation is key to normal development. Using statistics on many embryos, we will be able to study spatial patterns of mechanical properties. We will more specifically characterise how much of the patterning involved in brain development is due to variations in passive properties, and how the balance between active processes and the surrounding tissue is critical for normal development. Such questions and the methodology developed here to address them apply to most morphogenetic transformations in and are expected to be highly relevant elsewhere.